The homologous pairing and strand exchange activity purified from Ustilago maydis was the first example of a RecA-like activity to be discovered in eukaryotes. The gene encoding the protein responsible for the activity has now been identified and cloned. It is REC2, a gene necessary for DNA repair proficiency, efficient gene targeting during transformation, and meiosis. Analysis of the REC2 gene product has revealed some sequence homology with the Escherichia coli RecA protein. However, the sequence homology, which is limited to a particular region, and large size of the protein are features which set Rec2 protein apart from RecA and the RecA- like homologs that have been identified in eukaryotes. The Rec2 protein has been overproduced in E. coli and purified. The isolated protein is active in catalyzing DNA-dependent hydrolysis of ATP and the ATP-dependent homologous pairing reactions. We propose to investigate the pairing activity of the Rec2 protein in detail and to analyze the nature of Rec2- DNA complexes so as to understand the molecular mechanism of the homologous pairing reaction. Pairing reactions will be performed using a variety of oligonucleotides with structural perturbations. Conditions will be sought in which pairing between duplex oligonucleotides and homologous single-stranded DNA circles will proceed under ordinarily forbidden conditions. Previous studies have revealed that there is a duplex size threshold to pairing of 50-70 bp. Pairing proceeds efficiently if a duplex below that size threshold contains RNA. We are interested in discovering other structural means for activating pairing. Other studies to be performed include measuring unwinding of duplex induced in the presynaptic stage of reaction when the search for homology has not yet consummated. Pairing between polymorphic sequences will be examined in more detail. This will follow up on the finding that Rec2, like RecA, can pair DNA sequences of identical polarity and sequence. The nature of the paranemic joint will be studied using chemical reagents to probe for conformational changes induced in the DNA during pairing. DNA binding studies will be carried out to determine the stoichiometry of protein-DNA interactions. We will use techniques that yield binding information in real time as well as by end point determinations. Initial efforts will focus on measurements using surface plasmon resonance. We will try to correlate binding studies using spectroscopic means with some other means such as gel mobility shift. Such studies will provide us with more information on the nature of the DNA-protein complexes than has been possible using nitrocellulose filter retention methods. Site-directed mutants that have been previously characterized by genetic methods will be examined so as to gain insight into structure/function relationships.
| Bennett, R L; Holloman, W K (2001) A RecA homologue in Ustilago maydis that is distinct and evolutionarily distant from Rad51 actively promotes DNA pairing reactions in the absence of auxiliary factors. Biochemistry 40:2942-53 |
| Ferguson, D O; Rice, M C; Rendi, M H et al. (1997) Interaction between Ustilago maydis REC2 and RAD51 genes in DNA repair and mitotic recombination. Genetics 145:243-51 |
